The present invention generally relates to a process for enzymatic replacement of the C-terminal amino acid in the B-chain (B-30) of insulins from various species. In particular the invention relates to the conversion of porcine insulin to human insulin.
In earlier patent applications, e.g. U.S. application Ser. No. 364,856 filed Mar. 23, 1982 and based on International application No. PCT/DK81/00074 published on Feb. 4, 1982 under publication No. WO 82/00301 and European patent application No. 81303383.4 published on Feb. 3, 1982 under No. EP 45187 the applicant has described a general process for the replacement of the B-30 amino acid in insulins by reacting as substrate component the selected insulin with an amine component selected from amino acids, optionally substituted amino acids amides and amino acid esters in the presence of an L-specific serine or thiol carboxypeptidase enzyme in an aqueous solution or dispersion at pH 7 to 10.5. The preferred enzyme is carboxypeptidase Y (CPD-Y) from yeast (Saccharomyces cerevisiae) which may advantageously be used for converting porcine insulin (Ins'-Lys-Ala) to human insulin (Ins'-Lys-Thr) by means of a transpeptidation reaction with Thr-NH.sub.2.
Depending on the reaction conditions, especially the pH, this process leads directly to human insulin which may contain unreacted porcine insulin or to human insulin amide (Ins'-Lys-Thr-NH.sub.2) which may be separated from unreacted porcine insulin by HPLC and subsequently deamidated, preferably by means of CPD-Y. The overall yield in the latter case has been about 20-30%, it being understood that these figures refer to test runs and that optimalization of the reaction conditions has not been attempted.
Further details will appear from the above applications the whole contents of which are incorporated herein by reference.
Also the prior art is exhaustively discussed in the above-mentioned applications.
It has now surprisingly been found that the yields previously obtained according to the examples of the above-mentioned applications may be drastically improved by chemical modification of the L-specific serine carboxypeptidase enzymes provided certain reaction conditions are met.
Accordingly, the process according to the invention is characterized by
reacting as substrate component the selected insulin Ins-X, wherein X represents the B-30 amino acid,
with an amine component selected from the group consisting of
(a) L-amino acids of the formula EQU H--B--OH
wherein B is an L-amino acid residue,
(b) optionally N-substituted L-amino acid amides of the formula EQU H--B--NR.sup.1 R.sup.2
wherein B is an L-amino acid residue and R.sup.1 and R.sup.2 are independently selected from the group consisting of hydrogen, amino, hydroxy, alkyl, cycloalkyl, aryl, heteroaryl and aralkyl or R.sup.1 and R.sup.2 together with the nitrogen atom form a heterocyclic group which may contain a further hetero atom, and
(c) amino acid esters of the formula EQU H--B--OR.sup.3
wherein B is an L-amino acid residue and R.sup.3 represents alkyl, cycloalkly, aryl, heteroaryl or aralkyl,
in the presence of an L-specific serine carboxypeptidase enzyme which has been chemically modified at the sulfhydryl group by reaction with divalent metal ions in an aqueous solution or dispersion containing at least one of the following ions F.sup.-, Cl.sup.-, Br.sup.-, I.sup.-, CN.sup.-, SCN.sup.- and having a pH from about 5 to 10.5, thereby to form an insulin derivative.
Ins--B--OH, Ins--B--NR.sup.1 R.sup.2, Ins--B--B--NR.sup.1 R.sup.2 or Ins--B--OR.sup.3
or subsequently cleaving a group --NR.sup.1 R.sup.2, --B--NR.sup.1 R.sup.2 or --OR.sup.3, if desired.
In a preferred embodiment of the serine carboxypeptidase enzyme is treated with mercuric ions, preferably in the form of HgCl.sub.2 in the presence of a suitable buffer. It is a well established fact that carboxypeptidase Y and other serine carboxypeptidases contain an --SH (sulfhydryl) group in the form of a single cysteine residue, vide e.g. Hayashi et al., J. Biochem. 77, 1313-1318 (1975) (Ref. 1) and Bai et al., J. Biol. Chem. 254, 8473-8479 (1979) (Ref. 2) and Widmer et al. (Ref. 7), all being incorporated by reference.
Hayashi et al, investigated the effects of various metal ions on the peptidase and esterase activity of CPD-Y and found that preincubation with Cu.sup.++, Ag.sup.+, Hg.sup.++, Cu.sup.+, Mg.sup.++, Ca.sup.++, Ba.sup.++, Cr.sup.++, Mn.sup.++, Fe.sup.++ and Ni.sup.++ ion in amounts of 10.sup.-4 and 10.sup.-3 M resulted in significant losses of peptidase and esterase activities. A particularly significant loss was observed with Hg.sup.++ (added in the form of HgCl.sub.2) which inactivated the enzyme totally. Hayashi et al. assume that the inactivation as far as Hg.sup.++ is concerned is caused by a blocking of the --SH group.
Bai et al. has further investigated the properties of the --SH group and confirmed the significant decrease in peptidase activity for Hg.sup.++ -treated CPD-Y vs. native CPD-Y on most substrates.
On this basis it would not be expected that a carboxypeptidase Y chemically modified with a divalent metal ion, especially Hg.sup.++ (CPD-YM) would be capable of catalyzing the conversion of porcine insulin to human insulin amide by transpeptidation with threonine amide.
It was surprisingly found that the use of CPD-YM resulted in a drastic increase in the conversion yield up to 70 to 75% if only the reaction medium also contained halogen ions (F.sup.-, Cl.sup.-, Br.sup.- and I.sup.-) or pseudohalogen ions, e.g. CN.sup.- or SCN.sup.-.
Without wishing to be bound by any particular theory it is assumed that the halogen or pseudohalogen ions neutralize the positive charge on the CPD-YM thereby reactivating the inactivated enzyme. However, this mechanism does not explain the drastic yield increase, which will be further illustrated below by way of examples.
The applicable carboxypeptidases in the process of the invention are L-specific serine carboxypeptidases. Such enzymes can be produced by yeast fungi, or they may be of animal, vegetable or microbial origin.
A particularly expedient enzyme is carboxypeptidase Y from yeast fungi (CPD-Y). This enzyme is described in the earlier applications i.a. with reference to Johansen et al. (Ref. 4) who developed a particularly expedient purification method by affinity chromatography on an affinity resin comprising a polymeric resin matrix with coupled benzylsuccinyl groups. CPD-Y has the advantage of having no endopeptidase activity. It is available in large amounts and displays relatively great stability. Further details are given in Refs. 3 and 5.
In addition to CPD-Y, which is the preferred enzyme at present, the process of the invention is feasible with other carboxypeptidases, such as those listed in the following survey:
______________________________________ Origin Enzyme Fungi ______________________________________ Penicillocarboxypeptidase S-1 Penicillium janthinellum Penicillocarboxypeptidase S-2 Penicillium janthinellum Carboxypeptidase(s) from Aspergillus saitoi Carboxypeptidase(s) from Aspergillus oryzae Plants Carboxypeptidase(s) C Orange leaves Orange Peels Carboxypeptidase C.sub.N Citrus Natsudaidai Hayata Phaseolain French bean leaves Carboxypeptidase(s) from Germinating barley Germinating cotton plants Tomatoes Watermelons Bromelain(pineapple)powder ______________________________________
The close relationship between a number of the above carboxypeptidases is discussed by Kubota et al. (Ref. 6).
The process of the invention can in principle be carried out with any natural, semi-synthetic or synthetic insulin as substrate component.
The second participant in the reaction is the so-called amine component which is selected from the group consisting of
(a) L-amino acids of the formula EQU H--B--OH
wherein B is an L-amino acid residue,
(b) optionally N-substituted L-amino acid amides of the formula EQU H--B--NR.sup.1 R.sup.2
wherein B is an L-amino acid residue and R.sup.1 and R.sup.2 are independently selected from the group consisting of hydrogen, amino, hydroxy, alkyl, cycloalkyl, aryl, heteroaryl, and aralkyl or R.sup.1 and R.sup.2 together with the nitrogen atom form a heterocyclic group which may contain a further hetero atom, and
(c) amino acid esters of the formula EQU H--B--OR.sup.3
wherein B is an L-amino acid residue and R.sup.3 represents alkyl, cycloalkyl, aryl, heteroaryl or aralkyl.
The L-amino acid forming part of the amine component may be any of the known L-amino acids, e.g. leu, ile, ala, gly, ser, val, thr, lys, arg, asn, glu, gln, met, phe, tyr, trp or his.
In this context "alkyl" means straight chain or branched alkyl, preferably with 1 to 6 carbon atoms, e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert.butyl, amyl, hexyl and the like.
"Cycloalkyl" preferably means C.sub.3 -C.sub.8 cycloalkyl, e.g. cyclopropyl, cyclobutyl, etc.
"Aryl" is preferably phenyl and the like.
"Aralkyl" means benzyl, phenethyl, and the like. As stated the groups R.sup.1 and R.sup.2 may be the same or different.
"Heteroaryl" as well as the heterocyclic group which may be formed by R.sup.1, R.sup.2 and the nitrogen atom are represented by e.g. pyridyl, pyrrolidyl, pyrimidinyl, morpholinyl, pyrazinyl, imidazolyl, etc.
All of these groups may be substituted with substituents which are inert with relation to the enzyme, e.g. halo (fluoro, chloro, bromo, iodo), nitro, alkoxy (methoxy, ethoxy, etc.), or alkyl (methyl, ethyl, etc.).
Thus in case of all types of esters the group OR.sup.3 is preferably selected from alkoxy groups, such as methoxy, ethoxy or t-butoxy, phenyloxy, and benzyloxy groups. The groups may optionally be substituted with inert substituents, such as nitro groups (p-nitrobenzyloxy).
It is seen that in case of amides, when R.sup.1 =hydrogen, R.sup.2 =hydrogen represents the free amide, while R.sup.2 =OH is a hydroxamic acid, R.sup.2 =amino is a hydrazide, and R.sup.2 =phenyl represents an anilide.
As stated in claim 1, the process of the invention is carried out at pH 5.0 to 10.5, preferably at pH 7.0 to 8.5. The preferred pH-value, which is often within a very narrow range, depends upon the pH-optima and pH-minima, respectively, for the different enzymatic activities of the enzyme used.
It should be noted that the operative pH range is broader than when unmodified serine carboxypeptidases are used, since the modification leads to a reduced amidase activity in the range from pH 5 to pH 7, thereby making the isolation of an insulin amide possible even at this range.
If CPD-Y is used as the modified enzyme, the pH-value is preferably 7.0 to 8.5, which is particularly expedient, if an isolation of insulin amide intermediates is desired. At this range of the highest yields of insulin amide are obtained.
The selected pH-value should preferably be maintained throughout the coupling reaction, and may then be changed for precipitation of the reaction products, cleavage of protective groups, etc. A pH might be selected at which the enzyme displays a marked amidase activity whereby the desired insulin is formed in one step. However, preferably a pH is selected where the enzyme displays predominantly peptidase activity thereby favouring the formation of stable insulin amide intermediates, since these may easily be separated from unreacted insulin starting material.
pH-control may be provided by incorporating a suitable buffer for the selected pH-range in the reaction medium, such as a bicarbonate or HEPES buffer.
The pH-value may also be maintained by adding an acid, such as HCl, or a base, such as NaOH, during the reaction. This may conveniently be done by using a pH-stat.
Based on the information given above and in Ref. 3 and 5, the skilled person will be able to select the most suitable reaction conditions, especially with regard to the pH, by which the various enzymatic activities (amidase, peptidase, esterase, carboxypeptidase and peptidyl-amino-acid-amide hydrolase) might best be utilized depending upon the insulin substrate component, the amine component and the intention to suppress or favour the formation of intermediates.
Generally speaking low pH-values within the above range favour the formation and precipitation of an insulin amide intermediate, while higher values lead to a cleaving of the amide group due to the more pronounced amidase activity of the carboxypeptidase enzyme.
However, these conditions may also be influenced upon by varying the enzyme concentration, reaction time, etc.
The reaction is, as mentioned, carried out in a aqueous reaction medium which, if desired, may contain up to 50% by volume of an organic solvent. Preferred organic solvents are alkanols, e.g. methanol and ethanol, glycols, e.g. ethylene glycol or polyethylene glycols, dimethyl formamide, dimethyl sulfoxide, tetrahydrofurane, dioxane and dimethoxyethane.
The selection of the composition of the reaction medium depends particularly upon the solubility, temperature and pH of the reaction components and the insulin products involved and upon the stability of the enzyme.
The reaction medium may also comprise a component that renders the enzyme insoluble, but retains a considerable part of the enzyme activity, such as an ion exchange resin. Alternatively, the enzyme may be immobilized in a manner known per se, cf. Methods of Enzymology, Vol. 44, 1976, e.g. by bonding to a matrix, such as a cross-linked dextran or agarose, or to a silica, polyamide or cellulose, or by encapsulating in polyacrylamide, alginates or fibres. Besides, the enzyme may be modified by chemical means to improve its stability or enzymatic properties.
In case it is desired to suppress any precipitation of insulin amide intermediates, the reaction medium may also contain urea or guanidine hydrochloride in concentrations up to 6 molar. This may also be advantageous at pH-values nd in media where the insulin substrate component has a limited solubility.
The concentration of the two participants in the reaction may vary within wide limits, as explained below. A preferred starting concentration for the insulin substrate component is 0.002 to 0.05 molar and for the amine component 0.05 to 3 molar.
The enzyme concentration may vary as well, but the concentration is preferably 10.sup.-6 to 10.sup.-4 molar, in particular 10.sup.-5 molar. The most advantageous concentration depends i.a. on the substrate concentration, the amine concentration and the reaction time.
As earlier stated the presence of halogen ions (e.g. F.sup.-, Cl.sup.-, Br.sup.- or I.sup.-) or pseudohalogen ions (e.g. CN.sup.- or SCN.sup.-) in the reaction medium is decisive for the catalytic effect of the modified enzyme. Thus, the halogen ion concentration depends greatly on the enzyme concentration, at least stoichiometric amounts being necessary, but also on the composition of the reaction medium, the halogen ion in question etc. Generally speaking the concentration may vary from 10.sup.-4 molar to 2 molar.
According to the invention the reaction temperature is preferably 20.degree. to 40.degree. C. The most appropriate reaction temperature for a given synthesis can be determined by experiments, but depends particularly upon the used amine component and enzyme concentration. An appropriate temperature will usually be about 20.degree. to 35.degree. C., preferably about 30.degree. C. At temperatures lower than 20.degree. C. the reaction time will usually be inappropriately long, while temperatures above 40.degree. C. often cause problems with the stability of the enzyme and/or reactants or of the reaction products.
Similar variations occur for the reaction time which depends very much upon the other reaction parameters, especially the enzyme concentration. The standard reaction time in the process of the invention is about 2-6 hours.
It should be added that when using an amide or substituted amide as the amine component, it is normally desired to cleave the amide group specifically from the formed insulin amide. In this respect the unmodified carboxypeptidase, especially CPD-Y is very suitable since CPD-Y exhibits amidase activity at pH&gt;9 while the carboxypeptidase activity is negligible.
Also modified carboxypeptidase might be used, e.g. CPD-Y modified by methyl-Hg or ethyl-Hg. Cleaving with modified enzymes are preferably carried out at pH 7-10 at a temperature of 5.degree. to 35.degree. C.
By the same token the carboxypeptidase might generally be used to cleave the ester groups OR.sup.3, as defined from the formed insulin ester intermediate to obtain a final insulin which is not C-terminal protected.
Before the process of the invention will be illustrated by examples, starting materials, methods of measurement, etc. will be explained in general terms.
Starting materials
Porcine insulin was kindly supplied by Nordisk Insulin-laboratorium, Copenhagen. Both highly purified Zn-free insulin, Zn-insulin and crude insulin which had only been purified by citrate crystallization were used. Carboxypeptidase Y from baker's yeast, a commercial preparation of the Carlsberg Breweries, was isolated by a modification of the affinity chromatographic procedure of Johansen et al. (Ref. 4) and obtained as a lyophilized powder (10% enzyme in sodium citrate). Before use the enzyme was desalted on a "Sephadex G-25" column (1.5.times.25 cm), equilibrated and eluted with water. The concentration of the enzyme was determined spectrophotometrically using E.sub.280 nm.sup.1% =14.8. The enzyme preparation used was free of Protease A as checked by the assay of Lee and Riordan (Ref. 8). L-threonine amide as purchased from Vega-Fox, Arizona, USA. Chromatographic materials were products of Pharmacia, Sweden. All other reagents and solvents were analytical grade from Merck, W. Germany.
Amino Acid Analyses
Samples for amino acid analysis were hydrolyzed in 5.7M HCl at 110.degree. C. in vacuum for 24 hours, and analyzed on a Durrum D-500 amino acid analyzer.